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. 2022 Jan 18;132(2):e153357. doi: 10.1172/JCI153357

Figure 2. Desensitization of β3-AR depends on EPAC/RAP2A/PLC pathway activation.

Figure 2

(A) 3T3L1 adipocytes were pretreated with 10 μM ESI09 for 1 hour, then challenged with 10 μM CL-316243 for 3 hours (n = 3 per group, 3 independent experiments). (B) Active (GTP bound) RAP2 was determined by pulldown followed by Western blotting with RAP2 antibody (n = 2 per group). (C and D) FLAG-tagged RAP2A WT or constitutively active RAP2A (V12) was electroporated into 3T3L1 adipocytes (n = 1–2 per group, repeated once). (E and F) 3T3L1 adipocytes were pretreated for 1 hour with 10 μM U73122 or 50 μM BAPTA-AM, followed by 3 hours challenge with 10 μM CL-316243 (n = 3 per group, 3 independent experiments). (G) 3T3L1 adipocytes were pretreated with 10 μM ESI09 for 1 hour, challenged with 10 μM CL-316243, and calcium flux assessed in live cells using Fura2-AM (91 randomly chosen cells from 4 experiments [blue] and 101 from 3 experiments [gray]). Graph represents the subpopulation of cells that responded to CL-316243 (~20%). *Significance compared with control or GFP in all experiments. One-way ANOVA with Tukey’s post hoc comparisons (A, C, E, and F); independent samples t test (G). All error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.